A near-infrared fluorescent probe for rapid detection of carbon monoxide in living cells

Article abstract of DOI:10.1016/j.saa.2018.05.059

A near-infrared (NIR) and colorimetric fluorescent probe system was developed for Carbon Monoxide (CO) via a Pd⁰-mediated Tsuji-Trost reaction. In this probe, phenoxide anion formation (DPCO^?) was acted as the signal unit and an allyl carbonate group was used as the recognition unit. This non-fluorescent probe molecule can release the relevant fluorophore after conversion of Pd²⁺ to Pd⁰ by CO. The probe system including probe 1 and Pd²⁺ can be used for “naked-eye” detection of CO, and exhibited high selectivity to CO over various other sensing objects. More importantly, the probe system has great potential for fluorescence imaging of intracellular CO in living cells.

Full text of DOI:10.1016/j.saa.2018.05.059

Accepted Manuscript
A near-infrared fluorescent probe for rapid detection of carbon
monoxide in living cells
Liqiang Yan, Ding Nan, Cheng Lin, Yi Wan, Qiang Pan,
Zhengjian Qi
PII:
DOI:
Reference:
S1386-1425(18)30452-9
doi:10.1016/j.saa.2018.05.059
SAA 16093
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date: 26 December 2017
Revised date:
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date:
13 May 2018
15 May 2018
Please cite this article as: Liqiang Yan, Ding Nan, Cheng Lin, Yi Wan, Qiang Pan,
Zhengjian Qi , A near-infrared fluorescent probe for rapid detection of carbon monoxide
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authors. Please check if appropriate. Saa(2017), doi:10.1016/j.saa.2018.05.059
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ACCEPTED MANUSCRIPT
A near-infrared fluorescent probe for rapid detection of Carbon Monoxide in
living cells
＊
Liqiang Yana , Ding Nanb, Cheng Linb, Yi Wanb, Qiang Panb, Zhengjian Qib
＊
a College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, PR
China
b College of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu 211189,
PR China
Corresponding author: liqiangyan@glut.edu.cn (Liqiang Yan); qizhengjian506@163.com
(Zhengjian Qi)
Abstract:
A near-infrared (NIR) and colorimetric fluorescent probe system was developed for
Carbon Monoxide (CO) via a Pd0-mediated Tsuji-Trost reaction. In this probe,
phenoxide anion formation (DPCO-) was acted as the signal unit and an allyl
carbonate group was used as the recognition unit. This non-fluorescent probe
molecule can release the relevant fluorophore after conversion of Pd2+ to Pd0 by CO.
The probe system including probe 1 and Pd2+ can be used for “naked-eye” detection
of CO, and exhibited high selectivity to CO over various other sensing objects. More
importantly, the probe system has great potential for fluorescence imaging of
intracellular CO in living cells.
Keywords: Near-infrared, Fluorescence probe, Carbon Monoxide, Living cells
1. Introduction
In the human body and other biological systems, carbon monoxide (CO) can be
endogenously produced during the haem catabolism by heme oxygenase (HO)
enzymes, which is an important cell signaling molecule with substantial therapeutic
potential protecting from vascular, inflammatory, or even cancer diseases. [1, 2] More
importantly, it is now evident that CO can be used as a potential therapeutic agent
because of its reported antihypertensive, anti-inflammatory and cell-protective effects.
[3] Therefore, it is critically required to develop“smart” noninvasive imaging reagents
for the determination of CO in living systems.
In recent years, several analytical technologies have been developed to
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determination of CO in the chemical system, such as gas chromatography, [4]
chromogenic detection, [5-7] and electrochemical assays. [8] However, most of these
methods are effective for extracellular CO, while are not capable of precisely tracking
real-time CO in vivo in a noninvasive manner. Compared with the traditional
analytical technologies, fluorescence detection using fluorescent probes is more
attractive due to its superiorities of convenience, high sensitivity, and real-time and
nondestructive detection. [9-13] Up to now, several fluorescent probes have been
developed and applied for cell-imaging of CO. [14-20] These reported fluorescent
probes with absorption and emission in the blue–green light range have good
performance at the cellular level. However, the in vivo applications are quite limited
due to their poor capacity of resisting disturbance. One of the major obstacles
encountered with fluorescence imaging of CO in vivo is the strong intrinsic
autofluorescence background from living tissues, which significantly compromises
the accuracy of measurement under physiological conditions. [21, 22] Near-infrared
(NIR) fluorescent probes with long wavelength absorbance and emission (650–900
nm) are of considerable practical advantages due to decreased interference from
background autofluorescence, minimum photo-damage to biological samples, high
tissue depth penetration in the living systems. [23-29] Therefore, development of new
NIR fluorescent probes of CO with improved properties and reliable signal in living
cells is highly desired.
Herein, we developed a light-up NIR fluorescent probe system (probe 1 as shown
in scheme 1) for colorimetric and fluorescent detection of CO, in which the
chromophore DCPO is utilized as a NIR fluorescence reporter and an allyl carbonate
unit is utilized as the CO-active trigger. DCPO are widely used in fluorescence
imaging because of its NIR fluorescence and excellent light stability. [30, 31] The
strategy of this probe relies on the in situ conversion of Pd2+ to Pd0 by CO and the
well-known Pd0-mediated Tsuji−Trost reaction to remove the allyl carbonate group.
[32-34] The near infrared fluorescence Off-On switch is triggered by transformation
of allyl carbonate unit to phenol by the interaction with CO, which results in the
phenoxide anion formation (DPCO–). The probe system possesses several striking
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merits as follows: firstly, it exhibits near-zero background fluorescence but shows a
rapid, colorimetric, and remarkable NIR fluorescent turn-on response for CO, which
allows a convenient visual and sensitive fluorescent detection for CO; secondly, it can
be conveniently used to image both in vivo and in situ tracking of CO.
2. Experimental section
2.1. Materials and Instrumentation
All solvents and reagents used were analytical reagent and were used without further
purification unless specified. [Ru(CO)3Cl-(glycinate)] (CORM-3) is a commercially
available water-soluble CO-releasing molecule and can be used in the study as a CO
source. Silica gel (200–300 mesh) bought from Qingdao Ocean Chemicals was used
for column chromatography. Nitric oxide (NO) came from the aqueous solution of
1
sodium nitroferricyanide dihydrate (CAS: 1313755-38-9). H NMR and 13C NMR
spectra were recorded on a Bruker 600 MHz spectrometer using TMS as the internal
standard. UV-vis spectra were recorded on a UV-2450 UV/Vis spectrophotometer
(Shimadzu, Japan). Fluorescence spectra were measured with a fluorolog 3-TSCPC
spectrophotometer (Horiba Jobin Yvon Inc.). All measurements were prepared in
DMSO/PBS buffer solution (v:v=1:1, pH 7.4) and allowed to equilibrate at room
temperature for 40 min before spectral measurements.
2.2. Compound synthesis and characterization
2-(2-(4-Hydroxystyryl)-4H-chromen-4-ylidene)
malononitrile
(DCPO)
was
synthesized according to the procedure published in literatures as shown in Scheme 2.
[35, 36]
Synthesis of the Probe 1. To a solution of DCPO (312 mg, 1.0 mmol) in dry CH2Cl2
(10 mL) was added Et3N (0.5 mL) under N2 atmosphere. After stirring for 10 min, the
resulting red solution was cooled in an ice-bath, and then allyl chloroformate (1.04 g,
4 mmol) was slowly added over 10 min. During this time, the reacted mixture
changed to yellow and the solution was constantly stirred 14 hours at normal
temperature. After filtration, the crude product was precipitated out from the filtrate
under reduced pressure. The target product was afforded when the crude product was
further purified by column chromatography (dichloromethane/ethyl acetate = 10:1,
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1
v/v) as yellow solid (192.7 mg, 48.6%). m.p.168.3–170.1℃. H NMR (600 MHz,
DMSO-d6) δ (ppm): 8.75–8.74 (d, J=6.0 Hz, 1H), 8.01–7.99 (d, J=12.0 Hz, 1H),
7.96–7.93 (t, J = 9.0 Hz, 1H), 7.85–7.79 (m, 3H), 7.64–7.62 (t, J=6.0 Hz, 1H),
7.53–7.37 (m, 3H), 7.06 (s, 1H), 6.05–5.98 (m, 1H), 5.44–5.41 (d, J=18.0 Hz, 1H),
13
5.34–5.32 (d, J=12.0 Hz, 1H), 4.77–4.75 (t, J=6.0 Hz, 2H). C NMR (150 MHz,
DMSO-d6) δ (ppm): 190.79, 157.10, 155.51, 153.05, 152.29, 137.51, 134.75, 132.53,
131.28, 130.90, 129.09, 126.05, 125.84, 121.85, 121.73, 119.84, 119.09, 118.63,
115.58, 107.09, 69.43, 63.21.EI-MS calcd for C24H16N2O4 396.4, found 397.3[M+H]+.
2.3. Cell culture experiments
Breast cancer cells (MCF-7) were purchased from ATCC and were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal
Bovine Serum (FBS), 100 μg/mL penicillin and 100 μg/mL streptomycin in a 5% CO2,
water saturated incubator at 37℃, and then were seeded in a 24-well culture plate for
one night before cell imaging experiments. In the experiment of cell imaging, living
cells were incubated with probe 1 (5 μM) and a mixture of probe 1 (5 μM) + PdCl2
(10 μM) at 37 °C for 30 min, respectively. For imaging of exogenous CO, MCF-7
cells were pretreated with CO (40 μM) for 40 min at 37 °C and then were incubated
with a mixture of probe 1 (5 μM) + PdCl2 (10 μM) for 40 min at 37 °C. The imaging
of CO was then carried out upon excitation at 400 nm, emission window of red
channel and 20 objective lens.
The in vitro cytotoxicity of the probe system was studied with Alamar blue assay.
MCF-7 cells were seeded in 48-well plates at a density of 1  104 cells per well. After
24 h attachment, variant concentrations of probe 1 and PdCl2 were added and
incubated with the cells for another 24 h. Afterwards, the medium was removed and
each well was washed with PBS for three times. 250 mL Alamar blue solution was
added to incubate with cells. 4h later, 200 mL of this solution was pipetted from each
well and transferred into a 96-well plate for measurement. The absorbance was read at
570 nm (excitation)/600 nm (emission) using an automated microplate
spectrophotometer. The untreated cells were used as control and determined as 100%
viability. The blank Alamar blue without incubation with cells were taken as blank
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and used to calibrate the absorbance of each sample. The relative cell viability (%)
could be calculated by the ratio of the calibrated absorbance of a sample to the
calibrated absorbance of control group. Viable cells were stained green, while dead
cells were stained red.
3. Results and discussion
3.1. Colorimetric and Fluorescent Detection of CO
The absorption and fluorescence properties of the probe system (5 μM probe 1 + 10
μM PdCl2) were determined in PBS (pH =7.4) solution containing 50% of DMSO. As
shown in Fig. 1A, the probe system has a main absorption peaks at around 375 nm
and the solution of probe system shows light yellow. When the probe system was
treated with CO for 40 min, a new absorption peaks appeared at 575 nm with a
decrease of peak at 375 nm. The maximum absorption peak shifted from 375 nm to
575 nm. Along with increasing of CO concentration, the absorption titration curves of
the probe system increased gradually at 575 nm and the solution of the probe system
shows dark brown indicating the capacity of the probe system for colorimetric
detection of CO. As shown in Fig.1B, there was a good linearity between the
absorption intensity (A575) and concentration of CORM-3 ranging from 0 to 30 μM:
y=0.015+0.007[CO] (R2=0.9906).
To further identify the performance of the probe system, the emission spectra of the
probe was measured by fluorescence titration experiment. As shown in Fig. 2A, the
probe system showed almost no fluorescence emission upon excitation (λex) at 565 nm.
Addition of CO resulted in an obvious increase at 685 nm. With increasing
concentrations of CORM-3, the fluorescence titration spectrum showed a gradual
enhancement. As shown in Fig. 2B, there was a good linearity between the
fluorescence intensity at 685 nm and the concentration of CO ranging from 0 to 30μM
(Fig. 2B): y=3737.7+1202.7[CO] (R2=0.9980). The detection limit of CO was
calculated from the equation DL =3σ/S, where σ is the standard deviation of a blank
measurement and S is the slope between intensity versus sample concentration. The
detection limit of the probe system was calculated to be 5.710–8 M. As shown in Fig.
3A, upon addition of 40 μM of CORM-3, the probe system showed significant
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time-dependent fluorescent changes in the fluorescence spectra. Remarkably, the
fluorescence intensity of the probe solution at 685 nm reached nearly a maximum
value within 40 min as shown in Fig.3B. The time-dependent fluorescent change
measurements showed the response time of the probe system toward CO was 40 min
(Fig. 3).
3.2. CO selective and competitive experiment
The selectivity of the probe system was examined towards CO, various anions and
2−
2−
−
amino acids including F−, Cl−, Br−, I−, SO4 , HPO4 , HCO3 , Gly, Glu, Trp, Lys, Cys,
−
2−
GSH, HS−, ClO−, H2O2, NO2 , S2O3 , N2H4 and NO. As a result, only CO induced an
obvious turn-on fluorescence changes at 685 nm. There was little fluorescence
intensity increase when the probe system treated with other analytes (Fig. 4A). These
results clearly indicate that this probe system is highly selective for CO. Besides,
probe system had a good anti-interference for CO when mixing with others in
competitive experiment (Fig. 4B). These results indicated that the probe system has
reasonable selectivity to identify CO in a complex environment.
3.3. Fluorescence bio-imaging and cytotoxicity assay
On the basis of the excellent performances shown above, we investigated the potential
applications of the probe system for imaging of CO in living cells. As shown in Fig. 5,
when MCF-7 cells were incubated with probe 1 (5 μM) or the probe system (5 μM
probe 1 + 10 μM PdCl2) for 40 min, no fluorescence was observed (Fig.5b and Fig.5c).
However, when the cell culture was pre-incubated the probe system, then incubated
with CORM-3, intracellular red fluorescence was observed clearly (Fig.5d), indicating
the probe system is indeed an effective probe candidate for monitoring intracellular
CO changes in living cells.
To evaluate the cytotoxicity of the probe system, MCF-7 cells were incubated with
various concentrations of probe 1 and PdCl2 for 24 h. The cell viabilities were
evaluated via live/dead staining and Alarmar blue assay. As the live/dead staining
displayed, nearly all of the cells were stained green (live cells) at different probe
system concentrations (Fig.6a and 6b). As shown in Fig. 6c, even when the
concentration of the probe system was increased to as high as 15 μM for probe 1 and
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30 μM for PdCl2, stained green fluorescence (live cells) can be still observed.
However, most of the cells were showed red fluorescence (dead cells) when treated
with a high concentration of the probe system including 20 μM probe 1 and 40 μM
PdCl2 (Fig. 6d). The results of MTT assays also indicate that cell viability was over
85% when it is used below 15 μM probe 1 and 30 μM PdCl2 (Fig. 7). Therefore, these
results clearly indicate that the probe system possess low cytotoxicity to living cells.
3. Conclusion
In summary, a novel NIR fluorescent probe system of CO was prepared. It showed
admirable detection performances for CO with apparent colorimetric response,
evident fluorescence turn-on phenomenon, an ideal testing limit as low as 0.57 μM,
and low cytotoxicity to living cells. More importantly, this probe system has been
successfully applied to detect CO in living cells by laser confocal microscopy imaging.
Because of these excellent properties, this probe system possessed great potential for
practical applications in detection of CO in living systems.
Acknowledgements
This work was supported by Guangxi Key Laboratory of Electrochemical and
Magnetochemical Functional Materials (EMFM20181107), Policy Guidance Program
(Research Cooperation) Prospective Joint Research Project (BY2016076-02),
National Major Scientific Instruments and Equipment Development Projects
(2014YQ060773) and the Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (1107047002).
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Schemes and figures
Scheme 1. The structure and reaction mechanism of the probe 1
Scheme 2. Synthesis of probe 1. (a) Na, CH3COOC2H5, 51%; (b) H2SO4, CH3COOH,
62%; (c)malononitrile, AcOH, H2SO4, 14 h, 36.4%; (d) malononitrile, CH3COOOCH3,
25%; (e) Et3N，allyl chloroformate，CH2Cl2，48.6%。
Fig.1. (A) Absorption spectra of probe (5 μM probe 1 + 10 μM PdCl2) in the presence
of different concentration of CORM-3 in PBS buffer (10 mM, pH 7.4, with 50%
DMSO, v/v) at 37 °C, (B) a linear correlation between absorption intensity (A575) and
concentration of CORM-3.
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Fig.2. (A) Emission spectra (λex=565 nm) of probe (5 μM probe 1 + 10 μM PdCl2) in
the presence of different concentration of CORM-3 in PBS buffer (10 mM, pH 7.4,
with 50% DMSO, v/v) at 37 °C; (B) a linear correlation between emission intensity
and concentration of CORM-3 at 685 nm.
Fig.3. (A) Fluorescence spectra (λex=565 nm) changes of the probe (5 μM probe 1 +
10 μM PdCl2) upon addition of CORM-3 (40 μM) in PBS buffer (10 mM, pH 7.4,
with 50% DMSO, v/v) at 37 °C. (B) Fluorescent intensity changes at 685 nm as a
function of time.
Fig.4. (A) Fluorescence response of the probe system (5 μM probe 1 + 10 μM PdCl2)
to various analytes (40 μM) in PBS buffer (10 mM, pH 7.4, with 50% DMSO, v/v).
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(B) Fluorescence intensity at 685 nm of the probe system (5 μM probe 1 + 10 μM
PdCl2) in the presence of various analytes (40 μM). Black bars represent the addition
of 40 μM of the appropriate analytes to the probe system (5 μM probe 1 + 10 μM
PdCl2). Red bars represent the addition of the probe system (5 μM probe 1 + 10 μM
PdCl2) to the mixture solution of 40 μM the appropriate analytes and 40 μM CORM-3.
2−
2−
Analytes: (1) None, (2) CO, (3) F−, (4) Cl−, (5) Br−, (6) I−, (7) SO4 , (8) HPO4 , (9)
−
HCO3 , (10) Gly, (11) Glu, (12) Trp, (13) Lys, (14) Cys, (15) GSH, (16) HS−, (17)
−
ClO−, (18) H2O2, (19) NO2 , (20) S2O32− , (21) N2H4 and (22) NO.
Fig.5. Confocal microscope images of MCF-7 cells. (a) bright field image of untreated
cells; (b) cells treated with probe 1 (5μM) for 40 min; (c) cells treated with 5 μM
probe 1 and 10 μM PdCl2 for 40 min; (d) cells treated with probe 1 (5 μM) + PdCl2
(10 μM) and subsequent treatment with CORM-3 (10 μM) for 40 min. (b) (c) and (d)
images of cells upon excitation at 400 nm, emission window of red channel and 20
objective lens.
Fig.6. Cytotoxicity of probe 1. Fluorescence images of MCF-7 cells incubated with
variant concentrations of probe 1 and PdCl2 for 24 h: (a) 5 μM probe 1 + 10 μM PdCl2,
(b) 10 μM probe 1 + 20 μM PdCl2, (c) 15 μM probe 1 + 30 μM PdCl2 and (d) 20 μM
probe 1 + 40 μM PdCl2. The live cells were stained green, and the red indicates the
dead cells.
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Fig.7. Cell viability of MCF-7 cells after incubated with variant concentrations of
probe 1 and PdCl2 for 24 h. (a) 5 μM probe 1 + 10 μM PdCl2, (b) 10 μM probe 1 + 20
μM PdCl2, (c) 15 μM probe 1 + 30 μM PdCl2 and (d) 20 μM probe 1 + 40 μM PdCl2.
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Graphical Abstract
A near-infrared (NIR) and colorimetric fluorescent probe system was developed via a
Pd0-mediated Tsuji−Trost reaction, which showed colorimetric and excellent turn-on
responses for CO.
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Highlights
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Near-infrared (NIR) fluorescence emission.
Distinct colorimetric and excellent turn-on responses to CO.
Low cytotoxicity and bio-imaging in living cells.
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